Amido-1,2,3-thiadiazole derivatives as
novel S1P1 selective agonists
18th of September 2012
Nuria Aguilar
Multiple Sclerosis

Multiple Sclerosis is a chronic, inflammatory, demyelinating disease that affects the CNS.

Symptoms can vary widely depending on the part of the brain that is more affected. In
general: muscle weakness, abnormal muscle spasms, problems to speech, visual problems,
fatigue, chronic pain, changes in sensation…

It is considered as an autoimmune disease in which lymphocytes recognize myelin as a
foreign and attack it as if it were an invading virus.
Multiple Sclerosis

No definitive cause has been found. MS likely occurs as a result of some combination of
both enviromental and genetic factors.

In Europe, north of the 46th degree of latitude, MS, with an average of approximately 60
affected per 100.000 inhabitants, belongs to the most frequent neurological diseases.

MS primarily affects adults, with an age of onset typically between 20 and 40 years, and is
more common in women than in men.
Discovery of Fingolimod (FTY720)
HO2C
HO
NH2
OH
O
ISP-I, Myriocin
Potent immunosuppressant
isolated from Isaria sinclairii
OH
unnecessary
non-essential for activity
e.g. mycestericin D (4-deoxy)
and E (4-deoxy-3-epi-ISP-I)
NH2
OH
OH
Fingolimod
• Excellent immunosupressive activity
in vivo allograft models p.o.
• Improved toxicity profile
BMCL 1995, 5, 853 (Yoshitomi)
 In october 2010 Fingolimod, developed by Novartis, was aproved by the FDA and in most
european countries like the first oral treatment of MS under the tradename of Gylenia.
Fingolimod Mode of Action (I)
NH2
NH2
(S)
OH
SPHK2
OH
OPO3H2
OH
Fingolimod-phosphate
NH2
OPO 3H 2
(E)
OH
Sphingosine-1-phosphate
IC50 (nM) competition S133P binding
S1P1
S1P2
S1P3
S1P4
S1P5
0.47
0.31
0.17
95
0.61
metabolite
0.21
>105
5.0
5.9
0.59
FTY720
300
>105
>105
>5000
2623
S1P
S. Mandala et al. Science 2002 296, 346
Fingolimod-P is a non selective
full agonists of S1P receptors
Fingolimod Mode of Action (II)
 S1P, the endogenous ligand of S1P1 receptor, is involved in the migration of naïve lymphocytes
from lymph nodes (LN) to blood during immunosurveillance. Cells exit LN following the gradient
of S1P that exists between LN, lymph and blood. This effect is mediated by S1P1.
 Fingolimod-P and all synthetic S1P1
agonists cause S1P1 receptor
internalization, leading to disappearance of
the receptor from the cellular membrane
and making cells unable to respond to the
endogenous ligand.
 Thus, synthetic S1P1 agonists behave as
functional antagonists of the S1P1 receptor
and cause the retention of lymphocytes in
lymph nodes resulting in peripheral blood
lymphopenia.
 The retention of cells in the LN prevents
K. Powell, NIBR03_Feature_expert, www.nibr.com
autoreactive lymphocytes from migrating to
the disease target tissues (i.e., CNS) and
this results in clinical efficacy.
Almirall S1P1 Agonists Program Objectives
 Identification of a compound that does not require phosphorylation to be active
 Agonist of S1P1 and selective versus S1P3
S1P3: potential contribution to bradycardia, respiratory issues and blood
pressure, based on receptor expression pattern.
 Once a day oral compound with a DMPK profile able to:
- Prevent the exit of lymphocytes from LN to blood between administrations
- Permit fast recovery of lymphopenia after treatment discontinuation in
case of adverse events
- Direct PK/PD relationship (no hysteresis)
HTS Campaign: Identification of Amidothiadiazoles
1
2
EC50 S1P1(GTPS) = 4.5 M
EC50 S1P1(GTPS) = 9 M
Structural analysis of hit compounds:
S—O distance = 2.65 Å
O=C-N-C diedral angle = 2.5º
 Amidothiadiazole core forms a pseudo bicyclic system thanks to a S-O interaction
Comparison of ATDAs with known S1P1 agonists
Polar head
Novel lipophilic tail
Polar head
Linker
Linker
Lipophilic tail
1
Lipophilic tail
3*
 The additional lipophilic tail identified in our series resulted to be key for activity.
Its removal accounted for a complete lost in activity.
.* Vachal, P.; Toth, L. M.; Hale, J. J. Et al. Bioorg. Med. Chem Lett. 2006, 16, 3684-3687.
Initial analogues
Compound
S1P1 EC50
S1P3
%E@40µM
>10µM
-
1.5µM
31%
OMe
O
4
O
N
S
N
OH
N
F
O
5
O
N
S
N
OH
N
Initial analogues
Compound
S1P1 EC50
S1P3
%E@40µM
>10µM
-
1.5µM
31%
0.3 nM
300 nM
OMe
O
4
O
N
S
OH
N
N
F
O
5
O
N
S
N
OH
N
F
O
OH
O
6
N
S
N N
N
Homology model
 Docking of the initial analogues in an in-house generated homology model
based on bovine Rhodopsine
SAR conclusions
*
o-substitution preferred
Benzylic amides also allowed
*
N
H
N
O
N
H, Me low active
Propyl, CyMe potent
Bu optimal
S
*
COOH
COOH
O
OH
COOH
N
N
N N
Me led to increased S1P3
selectivity
 All analogues kept a reasonable S1P3 selectivity (>100 fold with examples in the
1000 fold selectivity range) and were stable in both human and rat liver microsomes
Bioorg Med Chem Lett accepted for publication
Compound 6: Lead profile
F
O
OH
O
N
N
S
N
Compound
S1P1
EC50(nM)
S1P3
EC50(nM)
S1P4
EC50(nM)
S1P5
EC50(nM)
6
0.3
300
106
62
N
Rat
microsomes
No Degr.
Human
microsomes
(% dgr.)
12%
8.2
4
Lymphopenia in Wistar rats
Dissability score
80
60
40
20
30
10
5
3
0
1
Lymphopenia (%)
100
-20
H.hepatocytes
Cl pred
(ml/min/Kg)
Dose (mg/kg)
Dose of Compound
6 (mg/Kg) po
Rat PK
Vss
(l/Kg)
Cl
(ml/min/Kg)
t1/2
(h)
F
(%)
2.0
16.6
1.8
50
Efficacy in the EAE rat model
3
2
1
0
8
10
12
14
Days after disease induction
Vehicle
Lymphopenia at 4h
Lymphopenia at 24h
Cpd 6_
10_
Cpd
1010mg/kg/qd
mg/Kg/qd po
16
Compound 6 elimination pathways
F
N
S
N
OH
N N
Compound 6
Taurine conjugate
100
90
80
70
60
50
40
30
20
10
0
O
1mg/kg iv single administration
LAS187940: %
dose dose
Compound
6:Total
% Total
O
Compound 6
LAS187940
Total: 80%
Amide hydrolisis
Total:7%
Feces
Urine
F
Feces
80%
O
Urine
7%
S
N
N
OH
N N
O
Compound 6: 1%
Compound 6: 45%
F
H
N
O
S
N
N
N N
OH
O
S
N
H
N
N N
O
S
O
O
OH
H
N
S
N
N N
OH
O
Amide Hydrolisis: 12%
Taurine conjugate: 23%
Amide Hydrolisis: 6%
 Compound 6 is eliminated in rats mostly unaltered and/or as a taurine conjugate.
 Amide hydrolisis is also observed but to a minor extent.
Compound 6 close analogues rat PK profile
Compound
S1P1
EC50 (nM)
S1P3
EC50 (nM)
Vss
(l/Kg)
Cl
(ml/min/Kg)
t1/2 (h)
2.3
900
3
49
1.4
0.9
860
0.7
4.8
2.2
1.4
1300
1
11.6
1.6
44
24%E at
10M
1.4
10
2.4
F
O
O
7
N
HN
S
N
OH
N
Cl
O
F
OH
O
8
N
S
N
N
N
F
O
9
OH
O
N
S
N
N
N
F
O
OH
O
10
N
S
N
N
N
Fenoxyethyl derivatives
F
O
N
S
N N
2
EC50 S1P1 = 9 M
11
EC50 S1P1 = 11 nM
cLogD(pH7.4) = 5.6
12
EC50 S1P1 = 2.4 nM
EC50 S1P3= 3.3 M
cLogD(pH 7.4)= 2.2
Met R/H: 8%/ <1%
t1/2 rat= 1.6 h
13
EC50 S1P1 = 16 nM
EC50 S1P3= 14 M
cLogD(pH 7.4)= 2.8
Met R/H: 6%/ 11%
t1/2 rat= 4 h
O
Compound 13: PK profile in rat and dog
LAS189040#1
1 mg/kg
i.v. Rat
plasma
levelslevels
Compound
13 at
1 mg/Kg
iv rat
plasma
700
Concentration (ng/ml)
600
AUC 0-∞
(ng*h/ml)
Vss
(l/kg)
Cl
(ml/min/kg)
t1/2 (h)
Rat
2653
2.1
6.3
4
Dog
435
1.5
38
0.7
Rat 1
500
Rat 2
400
300
Compd. 13
200
100
0
0
4
8
12
16
20
24
Tim e (h)
LAS189040#2
m g/kg
i.v. Dog
a levels levels
Compound
13 at1 1
mg/Kg
iv plasm
dog plasma
1000
Concentration (ng/ml)
900
800
Dog 1
700
Compd. 13
Human
microsomes
(% dgr.)
Plasma hydrolisis
at 5h
(%dgr.)
Hepatocytes
pred Cl
(ml/min/Kg)
Rat
Dog
Human
6
16
12
6%
6%
2%
6.3
33
19
Dog 2
600
500
400
300
•Compound 13 is strongly metabolized in both dog and human
hepatocytes, being the amide hydrolisis the major metabolite.
200
100
0
0
4
8
12
Tim e (h)
16
20
24
•Calculated Cl in hepatocytes does correlate very well with the
observed Cl in both dog and rat.
Compound 14: Hepatocytes studies
LAS189255
Metabolism en hepatocitos criopreservados
1.2
Cl
O
O
1.0
N
OH
S
N
Conc. (µM)
0.8
N N
O
0.6
0.4
0.2
0.0
0
30
60
Tim e (m in)
Rat
Dog
90
Human
120
Compd. 14
Hepatocytes
pred Cl
(ml/min/Kg)
In vivo Cl
(ml/min/Kg)
Rat
Dog
Human
3.9
3.4
4.0
5.5
1.5
-
 A change of a F by a Cl in the benzamide moiety accounts for a compound that is much less
metabolized in both human and dog hepatocytes.
 In dog hepatocytes amide hydrolisis is still the main metabolic pathway.
 In human hepatocytes no amide hydrolisis is seen. An oxidation metabolite is the major one.
Compd 14: Pharmacokinetic profile in two species
LAS189255
RatPK
PK(1(1mg/Kg
mg/kgi.v.)
i.v.)
Compound
14 Rat
10000
Rat 1
Concentration (ng/ml)
Concentration (ng/ml)
1000
LAS189255
Compound
14Dog
Dog PK
PK (1
(1 mg/kg
mg/Kg i.v.)
i.v.)
Rat 2
100
10
1
Dog 1
Dog 2
1000
100
10
1
0
4
8
12
16
20
24
28
0
8
16
24
32
Time (h)
40
48
56
64
72
80
Time (h)
Cmax
(ng/ml)
AUC last
(ng*h/ml)
AUC 0-∞
(ng*h/ml)
Cl
(ml/min/kg)
Vz
(l/kg)
Vss
(l/kg)
MRT
(h)
t1/2
(h)
Rat
690
2995
3040
5.5
1.9
1.9
5.7
4.0
Dog
1193
11077
11351
1.5
1.6
1.5
17
12.5
 Half life prediction to humans with two species indicates that the compound could likely
be dosed once a day (22h expected half life).
Compound 14: Pre-candidate profile
Cl
O
O
N
OH
S
N
N N
O
Compound
S1P1
EC50(nM)
S1P3
EC50(nM)
S1P4
EC50(nM)
S1P5
EC50(nM)
14
5.1
4000
120
25
EAE 02_09
LAS189255 Daily dose
Lymphopenia in rat and dog
Efficacy in the EAE rat model
4
RAT
DOG
80
60
40
20
0
Clinical score
Lymphopenia (%)
100
3
2
1
24
h
54
h
4h
24
h
4h
4h
24
h
0
8
10
12
14
16
18
Time (days)
3mg/kg
10mg/kg
3mg/kg
LAS189255 po
Compound
14 po



Vehicle
Vehicle
LAS 189255 3mg/kg
Compound
14 at 3 mg/Kg
Compound
14 at 10 mg/Kg
LAS 189255 10mg/kg
Rat bradycardia <15% at 100 mg/Kg
No ADME in vitro liabilities found (CYP450 inhibition nor Glutation adduct formation)
No hERG activity observed
Mechanism of action: Chemotaxis Assay
Chemotaxis induced by S1P in Murine spleen lymphocytes_ Assay principle
A) Migration in presence of Compound
+ Compd
Splenocytes
S1P 10nM
B) 30’ Preincubation with Compnd
S1P like
Fingo like
+ Cmpnd
Wash
out
Migration in absence of Compnd
S1P 10nM
S1P 10nM
Chemotaxis assay results and translation
in vivo
Chemotaxis induced by S1P 10nM
in murine splenocytes
Compound 14
Fingolimod
Migration inhibition (%)
Migration inhibition (%)
150
100
50
0
-12
-10
-8
-6
-4
Log [FTY720] (M)
100
50
0
-12
-50
-10
-8
-6
-4
Log [189255]
(M) (M)
Log
(Cpd. 10)
Continuous
30min / wash out
 Fingolimod interaction with S1P1 induce receptor internalization and slow recycling.
 In Compound 14 the recycling is fast and continuous presence of the compound is need for
efficay.
PK / PD LAS189255
1000
75
750
50
500
25
250
0
12
24
Time (h)
36
48
0
Plasma levels (ng/ml)
Lymphopenia (%)
100
Compound
3mg/kg 14
po
Ly
PKPD
05_09
vs LyPKPD
Lymphopenia
in06_09
rats
 Lymphopenic effect of compound 14 only depends
on plasma levels and the time to recover from
lymphopenia after treatment stop can be predicted
from farmacokinetics
Summary and conclusions
 A novel structural class of S1P1 agonists that do not require phosphorylation
to be active has been identified.
 These compounds are potent agonists of S1P1 and selective versus S1P3.
 After an intensive optimization process Compound 14 has been identified as a
promising pre-candidate suitable for once a day administration.
 Its mechanism of action suggests a direct PK/PD relationship.
The Medicinal Chemistry Team